The present invention relates to Drosophila genes and methods for their use. The invention provides nucleotide sequences of Drosophila genes, amino acid sequences of the encoded proteins, and derivatives (e.g., fragments) and analogs thereof. The invention further relates to fragments (and derivatives and analogs thereof) of proteins which comprise one or more domains of a Drosophila protein. Antibodies to Drosophila proteins, and derivatives and analogs thereof, are also provided. Also provided herein are vectors and host cells comprising such nucleic acids. Methods of production of a Drosophila protein (e.g., by recombinant means), and derivatives and analogs thereof, are provided. Chimeric polypeptide molecules comprising polypeptides of the invention fused to heterologous polypeptide sequences are provided. Methods to identify the biological function of a Drosophila gene are provided, including various methods for the functional modification (e.g., overexpression, underexpression, mutation, knock-out) of one gene, or of two or more genes simultaneously. Methods to identify a Drosophila gene which modifies the function of, and/or functions in a downstream pathway from, another gene are provided. The invention further provides for use of Drosophila proteins as media additives or pesticides.
Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.
G-protein coupled receptors (GPCRs) form an extensive family of transmembrane regulatory proteins that elicit intracellular signals in nearly every physiological system of chordates and invertebrate organisms. As a consequence of relatively small ligand-binding sites and the wide range of physiological events which they regulate, GPCRs have established the precedent of being the largest class of drug targets in humans. As described below, the sequence conservation of GPCRs across vertebrate and invertebrate species suggests that novel receptors identified in invertebrate species could aid in identification of homologues in other species, and the application of the appropriate agonist or antagonist mammalian receptor drugs to invertebrate species could result in the identification of effective pesticide agents. GPCRs can be divided into five broad structural classes, A-E, based on amino acid sequence similarity and sequence motifs. The largest class is class A, which can, in turn, be divided into subgroups according to receptor sequence similarity and ligand characteristics. The categorization of these relationships is illustrated by the following examples:
Class A (rhodopsin-like) GPCRs include: biogenic amine receptors (e.g. xcex1-adrenergic, xcex2-adrenergic, dopamine, histamine, muscarinic acetylcholine, melatonin, 5-HT, octopamine and tyramine); peptidic ligand receptors (e.g., angiotensin, bombesin, chemokine, endothelin, galanin, hormone protein, F-met-leu-phe, melanocortin, N-formyl peptide, neuropeptide Y, neurokinin, opiate, tachykinin, vasopressin, oxytocin and somatostatin); rhodopsin receptors (e.g., vertebrate rhodopsin, arthropod rhodopsin, and olfactory receptors); prostanoid receptors (e.g., prostaglandin, prostacyclin, and thromboxane); nucleotide receptors (e.g., adenosine and purinoceptors); hormone-releasing GPCRs (e.g., gonadotropin-releasing hormone, thyrotropin-releasing hormone, growth hormone, and secretagogue GPCRs);
Class B (secretin like) GPCRs include calcitonin, calcitonin releasing factor, calcitonin gene-related peptide, gastrin, cholecystokinin, glucagon, growth hormone-releasing hormone, parathyroid hormone, vasoactive intestinal peptide, PACAP, diuretic hormone and secretin GPCRs;
Class C (metabotropic glutamate-like) GPCRs include metabotropic glutamate, metabotropic GABAB, and extracellular calcium-sensing GPCRs;
Class D includes pheromone GPCRs; and
Class E includes cAMP-binding GPCRs.
Among their many functions, extensive study has revealed that GPCRs play a prominent role as receptors for neurotransmitters within the central and peripheral nervous systems, notably illustrated by the biogenic amine ligands such as norepinephrine (NE), octopamine (Oct), dopamine (DA), acetylcholine (ACh) and 5-hydroxytryptamine (5-HT). Several GPCRs for the biogenic amines have been identified in insects by pharmacological and molecular cloning approaches, including two octopamine/tyramine receptors from Drosophila melanogaster (Arakawa et al., 1990, Neuron 4, 343-354; Saudou et al., 1990, EMBO 9, 3611-3617; Han et al., 1990, J. Neurosci. 18, 3650-3658; see also Venter et al, U.S. Pat. Nos. 5,474,898 and 5,344,776) as well from other insect species such as moth, locus, and honey bee (von Nickisch-Rosenegk et al., 1996, Insect Biochem. Mol. Biol. 26: 817-827; Hiripi et al., 1994, Brain Res. 7, 119-126; Roeder et al., 1995, Prog. Brain Res. 106, 249-258; Evans, 1987, J. Exp. Biol. 129:239-250; Ebert et al., 1998, Insect Mol. Biol. 7, 151-162). Two putative Drosophila dopamine receptors, (Sugamori et al., 1995, FEBS Lett. 362, 131-138; Feng et al., 1996, J. Neurosci. 16, 3925-3933), two 5-HT receptors (Saudou et al., 1992, EMBO J. 11, 7-17), and one muscarinic acetylcholine receptor (Shapiro et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86, 9039-9043) have been identified and molecularly cloned, and are shown to be expressed in the nervous system. Glutamate and GABA are also CNS neurotransmitters involved in memory and mediation of pain sensation, and the metabotropic glutamate and GABA GPCRs form a structurally separate class of receptors (Class C). A Drosophila metabotropic glutamate receptor has been cloned and is expressed in the embryonic central nervous system (CNS) (Parmentier et al., 1996, J. Neurosci. 16, 6687-6694).
Other classes of GPCRs, such as rhodopsins and odorant receptors, are light and chemosensory receptors for the CNS and enable the senses of vision and odor. A number of rhodopsin genes have been identified and sequenced from Drosophila melanogaster, virilis, and simulans (Carulli et al., 1994, J. Mol. Evol. 38, 250-262), honey bee (Bellingham et al., 1997, Eur. J. Biochem. 243, 775-781; Townson et al., 1998, J. Neurosci. 18, 2412-2422), ant and tobacco hornworm (Chase et al., 1997, J. Exp. Biol. 200, 2469-2478; Popp et al., 1996, Invert. Neurosci. 1, 323-329). Related to actions within the nervous system, GPCRs regulate aspects of neuroendocrine secretion such as thyroid releasing hormone (TRH), thyroid stimulating hormone (TSH), growth hormone releasing hormone (GHRH), adrenocorticotropin (ACTH), and water balance via diuretic hormone or vasopressin. Extending to the regulation of cell growth and mammalian reproduction, GPCRs can couple to mitogenic pathways as illustrated by the bombesin and endothelin receptors, and can control ovulation, lactation and birth through the action of follicular stimulating hormone (FSH), luteinizing hormone (LH), and oxytocin.
Numerous neuropeptides have been identified from insects which perform similar overall roles to some of these mammalian peptides. However, the molecular characterization is incomplete with respect to the insect GPCRs which bind these analogous peptides; several may be homologous to mammalian receptors. A number of diuretic peptides, including an arginine vasopressin-like diuretic hormone, have been identified in locusts (Lehmberg et al., 1991, Biochem. Biophys. Res. Comm. 179, 1036-1041; Thompson et al., 1995, Peptides 16, 95-104; Schoofs et al., 1997, Peptides 18, 145-156; Proux et al., 1987, Biochem. Biophys. Res. Comm. 149, 180-186), while a diuretic hormone GPCR has been cloned from the house cricket and tobacco hornworm (Reagan, 1996, Insect Biochem. Mol. Biol. 26, 1-6; Reagan, 1994, Biol. Chem. 269, 9-12). A Drosophila GPCR with 50% identity to the transmembrane regions of rat TSH, FSH, and LH receptors has been cloned and sequenced (Hauser et al., 1997, J. Biol. Chem. 272, 1002-1010). Within the immune system, GPCRs can influence neutrophil chemotaxis through the action of chemokines, while modulation of digestion via gastric secretion and gut motility is regulated through GPCRs by the peptidic ligands gastrin and cholecystokinin (CCK). Insect GPCRs related to chemokine or CCK receptors have not been previously reported. However, leucosulfakin, proctolin, and FMRFamide peptides, which share sequence identity or functions similar to CCK/gastrin, have been isolated from several insect species (Nachman et al., 1986, Science 234, 71-73; Veenstra, 1989, Neuropeptides 14, 145-149; De Loof and Schoofs, 1990, Comp. Biochem. Physiol. [B] 95, 459-468; Starrat and Brown, 1975, Life Sci. 17, 1253-1256; O""Shea and Adams, 1981, Science 213, 567-569), including Drosophila (Nichols et al., 1988, J. Biol. Chem. 263, 12167-12170). These examples suggest a general structural conservation of certain GPCR subclasses across vertebrate and invertebrate species, and reinforce the notion that GPCRs play essential roles in the signaling and regulation of invertebrate physiology, such as for insects.
Gamma aminobutyric acid (GABA) is an important inhibitory neurotransmitter in both insects and vertebrates (Kuffler et al., 1965, Neurophysiol. 21, 589-601; Usherwood et al., 1965, Neurophysiol. 28, 497-519). In vertebrates, there are three broad families of GABA receptors found on both pre- and post-synaptic membranes. The GABAA and GABAC families are ionotropic receptors, i.e., ligand-gated ion channels which mediate fast inhibitory neurotransmission (Hevers et al., 1998, Mol. Neurobiol. 18:35-86; Bormann et al., 1995, Trends Neurosci. 18:515-519). Homologs of these ionotropic receptors are known in insects. For example, the receptor rdl belongs to the GABAA class, and is the target of cyclodiene pesticides such as Dieldrin (ffrench-Constant et al., 1993, Naure 363, 449-451).
The GABAB family of vertebrate receptors are entirely distinct from the GABAA and GABAC classes in structure and mechanism; they are seven transmembrane domain G-protein coupled receptors (GPCRs) which mediate slow, long-lasting inhibitory effects of GABA via G-mediated effects on the activity of adenylate cyclase, and potassium and calcium channels (for review, see Bowery, 1993, GABAB receptor pharmacology, Annu. Rev. Pharmacol. Toxicol. 33, 109-147; Kerr and Ong, 1995, GABAB receptors, Pharmacol. Ther. 67, 187-246). Initial cloning of the vertebrate GABABR1 receptor (Kaupmann et al., 1997, Nature 386, 239-246) suggested that additional subunits may be required to mediate the full physiologic response to GABA. Subsequent work has identified a second subunit, GABABR2, which physically associates with GABABR1 to form an unusual heteromeric GPCR that appears to mediate the complete physiologic effects of GABAB receptors (Kuner et al., 1999, Science 283, 74-77; Jones et al., 1999, Nature 396, 674-678; White et al., 1999, Nature 396, 679-682; and Kaupmann et al., 1999, Nature 396, 683-687).
Vertebrate GABAB receptors are activated specifically by baclofen and 3-aminopropylphosphonous acid (3-APPA) and are blocked by phaclofen, saclofen and CGP35348 (Bowery, 1993, GABAB receptor pharmacology, Annu. Rev. Pharmacol. Toxicol. 33, 109-147). However, baclofen is inactive in many invertebrate preparations, leading investigators to question if this receptor class existed in invertebrates (Sattelle et al., 1988, GABA receptors on the cell body membrane of an identified insect motor neuron, Proc. R. Soc. Lond. B 232, 445-456; Benson, 1989, A novel GABA receptor in the heart of a primitive arthropod, Exp. Biol. 147, 421-438 ). Furthermore, photoaffinity crosslinking studies with the ligand CGP71782, did not reveal the presence of GABAB receptors in invertebrates, though receptors from several vertebrate species were identified (Kaupmann et al., 1997, Nature 386, 239).
The biogenic amines form one of the largest subgroups of neurotransmitters, and regulate numerous physiological processes. (see Goodman and Gilman""s: The pharmacological basis of therapeutics, ninth edition, p. 118-130, Hardman and Limbird, eds., McGraw-Hill, New York). One of the most widely characterized biogenic amine ligand and receptor systems are those of the catecholamines. These include the adrenergic system (xcex1-adrenergic and xcex2-adrenergic), which is central to autonomic functions such as sympathetic regulation of arteriole smooth muscle contraction and dilatation, and to regulation of heart contractility and conduction velocity. Epineprine and norepinephrine are the physiological adrenergic ligands; they signal their action through xcex2- and xcex1-adrenergic GPCRs. Another catecholamine is dopamine, which can also influence vascular smooth muscle contractility predominately through the action of receptors in the renal, coronary and mesenteric arteriole beds. In the CNS, dopamine plays a critical role in initiating voluntary motor movement through post-synapatic stimulation of the extrapyramidal motor system. Deficits in this pathway are most notable with the uncontrolled tremors observed in human Parkinson""s disease. Octopamine is a related biogenic amine whose role in mammals is not clear, but which plays a significant role in invertebrates as a neurotransmitter and hormone (Evans, 1980, Adv. Insect Physiol. 15, 317-473), and acts similarly through a class of GPCRs closely related to the mammalian adrenergic receptors (Arakawa et al., 1990, Neuron 4, 343-354; Evans et al., 1993, Neurochem. Res. 18, 869-874).
In mammals, the actions of epinephrine and norepinephrine are mediated through three xcex2-adrenergic receptors (xcex21, xcex22 and xcex23) and six xcex1-adrenergic receptors (xcex11A, xcex11B, xcex11D, xcex12A, xcex12B and xcex12C) (see Kobilka, 1992, Annu. Rev. Neurosci. 15, 87-114; Insel, 1993, Exp. Gerontol. 28, 341-348; Hein et al.,1995, Neuropharmacology 34, 357-366). These seven transmembrane-spanning domain GPCRs are charcterized by a large intracellular loop connecting transmembrane domains five and six; this intracellular loop domain contributes to the interaction with specific heterotrimeric G-proteins which mediate downstream second messenger signaling events (Kobilka et al., 1988, Science 240, 1310-1316).
The xcex2-adrenergic receptors can be distinguished pharmacologically from the xcex1-adrenergic receptors by the use of subtype-specific agonists and antagonists (see Lefkowitz, 1979, Ann. Intern. Med. 91, 450-458; Frielle et al., 1989, Clin. Chem. 35, 721-725; Ruffolo et al., 1995, J. Med. Chem. 38, 3681-3716; and Kobilka et al., 1988, Science 240, 1310-1316). The intracellular effects of ligand-dependent activation of xcex2-adrenergic receptors result principally from the intracellular increase in cAMP via adenylyl cyclases (see Lefkowitz, 1976, In: Properties of purified cholinergic and adrenergic receptors, pp.69-83; Strosberg, 1995, Obes. Res. 4, 501S-505S). Subtype diversity of xcex1-adrenergic receptors is correlated to the intracellular signaling through multiple but specific effector pathways. These include IP3 and Ca+2 mobilization, diacylglycerol (xcex11A-D), activation of adenylyl cyclase, decreased cAMP, increased K+ conductance, and decreased Ca+2 conductance (xcex12A-C) (Kobilka, 1992, Annu. Rev. Neursci. 15:87-114; Hein et al., 1995, Neuropharmacology 34, 357-366).
Diversity also exists within dopamine GPCR signaling. Five dopamine receptor subtypes (D1-5) have been pharmacologically and molecularly characterized in mammals as being structurally similar to the adrenergic receptors. Subtype-specific agonists and antagonists are available for several of the dopamine receptors, notably for D1-3. The dopamine receptors couple to a similar intracellular signaling pathway as do the adrenergic receptors; D1 receptor elicts the activation of adenylyl cyclase while the D2 receptor is coupled to the decrease of cAMP, reduced Ca+2 conductance and increased K+ conductance. Although there have been reports of mammalian octopamine receptors with pharmacological properties distinct from adrenergic and dopamine receptors (Hicks et al., 1979, Brain Res. 157, 402-406), and octopamine exists in mammalian brain (see Axelrod et al., 1977, Nature 265, 501-504), these receptors have not been molecularly identified.
Catecholamine signaling in invertebrates has, to date, been demonstrated by the existence of dopamine-elicited physiology and GPCRs sharing similarity to mammalian GPCRs. Two dopamine GPCRs have been identified and cloned from Drosophila, (Sugamori et al., 1995, FEBS Lett. 362, 131-138; Feng et al., 1996, J. Neurosci. 16, 3925-3933), and share pharmacological and intracellular signaling characteristics of mammalian D1 and D2 receptors (Sugamori et al., 1995, FEBS Lett. 362, 131-138; Feng et al., 1996, 16, 3925-3933; Yellman et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94, 4131-4136; Reale et al., 1997, J. Neurosci. 17, 6545-6553; Torres et al., 1998, Synapse 29, 148-161). However, the pharmacological profile of the insect D1-like receptor is not totally consistent with the profile exhibited by the mammalian receptor (Sugamori, 1995, FEBS Lett. 362, 131-138). Epinephrine and norepinephrine have been identified in invertebrates and can elict invertebrate biological responses (see Murdock, 1971, Comp. Gen. Pharmacol. 7, 254-274 for review; Watson et al., 1993, J. Pharm. Biomed. Anal. 11-12, 1145-1149; Johnson et al., 1997, J. Comp. Physiol. [B] 167, 89-97; Park et al., 1998, Gen. Comp. Endocrinol. 110, 88-95). However, the physiological receptors which mediate the action of these catecholamines in invertebrates is not clear. The GPCRs most highly related to the adrenergic receptors in invertebrates are the three octopamine receptors identified in multiple insect species (OCT1-3). The insect octopamine receptors are pharmacologically similar to mammalian xcex1-adrenergic receptors (Evans, 1981, J. Physiol. 318, 99-122; Evans, 1987, J. Exp. Biol. 129, 239-250; Venter et al., 1988, Biochem. Pharmacol. 38, 1197-1208; Nathanson, 1993, Pharmacol. Exp. Ther. 265, 509-515; see also Evans et al., 1993, Neurochem Res. 18, 869-874). However, there can be considerable cross-interaction of mammalian dopaminergic and adrenergic receptor agonists and antagonists with the invertebrate octopamine and related tyramine receptors, resulting in difficulties in assigning the specific signaling role for a given invertebrate biogenic amine receptor.
Accordingly, there exists a need in the art to determine the identity and role of GPCRs in invertebrates, including flies and other insects. Such determinations may lead to the development of powerful and specific pesticides, and further, may enhance understanding of mammalian, including human, physiologic functions of this important superfamily of receptor molecules. This invention provides novel fly GPCRs and methods for their use, as described in detail below.
Knowledge of the expressed and/or genomic sequences of an organism is a great aid to the use of that organism as a research tool in the study of genes and their function. Drosophila melanogaster is a model system that enables powerful genetic manipulations and studies not available in higher organisms, and thus knowledge of its expressed sequences is highly desired. The present invention also provides such nucleotide and protein sequences of D. melanogaster genes.
The present invention relates to full-length and partial nucleotide sequences. of D. melanogaster genes, including full-length fly GPCR genes, amino acid sequences of the encoded proteins, and derivatives (e.g., fragments) and analogs thereof. Nucleic acids capable of hybridizing to or complementary to the foregoing nucleotide sequences are also provided. The invention further relates to a method of identifying genes that are modified by, or that participate in signal transduction with, D. melanogaster GPCR genes. The invention still further relates to derivatives and analogs of D. melanogaster GPCR genes and proteins which are functionally active (e.g., xe2x80x9cminigenesxe2x80x9d), and to D. melanogaster protein fragments which are capable of displaying one or more known functional activities associated with a full-length (wild-type) GPCR protein. Such functional activities include but are not limited to antigenicity (ability to bind to, or to compete for binding with, an anti-peptide antibody), immunogenicity (ability to generate antibody), and ability to bind to (or to compete for binding with) a GPCR ligand. The invention further relates to a fragment (or a derivative or analog thereof) of a D. melanogaster GPCR protein which comprises one or more domains of the protein, such as a transmembrane domain, a ligand-binding domain, or a cytosolic domain (e.g., the intracellular loop domain between transmembrane domains five and six of the GPCR). Antibodies to D. melanogaster GPCR proteins, and derivatives and analogs thereof, are additionally provided. Methods of production of such proteins, derivatives and analogs, e.g., by recombinant means, are also provided. Methods to identify the biological function of a Drosophila GPCR gene are provided, including various methods for the functional modification (e.g., overexpression, underexpression, mutation, knock-out) of one gene, or of two or more genes simultaneously. Methods to identify a Drosophila gene which modifies the function of, and/or functions in an upstream or downstream pathway from, a D. melanogaster GPCR gene are provided. The invention further provides for use of Drosophila GPCR proteins, derivatives, fragments, or ligands thereof as media additives or pesticides.
This invention provides a method of detecting the effect of expression of a D. melanogaster GPCR gene which encodes a D. melanogaster GPCR protein comprising an amino acid sequence as set forth in FIG. 1 (SEQ ID NO:2), FIG. 2 (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6), or FIG. 4 (SEQ ID NO:32,370), on a D. melanogaster signaling pathway, the method comprising: (a) mutating or abnormally expressing a wild-type D. melanogaster GPCR gene that encodes a protein comprising an amino acid sequence as set forth in FIG. 1 (SEQ ID NO:2), FIG. 2 (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6), or FIG. 4 (SEQ ID NO:32,370), in a fly already having a mutation in a D. melanogaster signaling pathway that displays a phenotype-of-interest; and (b) detecting the effect of step (a) on the phenotype-of-interest, so as to detect the effect of expression of the D. melanogaster GPCR gene.
This invention provides a cell culture medium or medium supplement comprising: (a) a sterile liquid carrier; and (b) a protein encoded by a first nucleic acid which hybridizes under conditions selected from the group consisting of high stringency, moderate stringency and low stringency to a second nucleic acid, which second nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:741. In another embodiment, the nucleotide sequence is selected from the group consisting SEQ ID NO:15,289 through SEQ ID NO:31,635.
The invention provides a purified Drosophila melanogaster G-protein coupled receptor (xe2x80x9cGPCRxe2x80x9d) comprising or defined by the amino acid sequence set forth in SEQ ID NO:2, 4, 6 or 32,370, as well as purified derivatives thereof which are able to display one or more functional activities of a Drosophila melanogaster GPCR protein. In particular embodiments, the derivative is able to be bound by an antibody directed against a Drosophila melanogaster GPCR protein, or is a fragment.
The invention also provides a purified fragment of a Drosophila melanogaster GPCR, which fragment comprises a domain of the GPCR protein selected from the group consisting of the extracellular domain, the intracellular domain, a membrane spanning domain, and the ligand-binding domain. In a particular embodiment, the fragment comprises at least one membrane spanning domain.
The invention also provides a mature Drosophila melanogaster GPCR protein defined by an amino acid sequence of SEQ ID NO:2, 4, 6 or 32,370 from which the secretory signal peptide sequence has been removed.
The invention further provides a purified protein comprising at least 25 contiguous amino acids of the sequence set forth in any one of SEQ ID NO:2, 4, 6 or 32,370. In a specific embodiment, such protein is fused to a second protein that is not a GPCR protein. The invention further relates to a chimeric protein comprising a fragment of a Drosophila melanogaster GPCR protein consisting of at least 25 contiguous amino acids of the an amino acid sequence set forth in SEQ ID NO:2, 4, 6 or 32,370 fused to a second protein, in which the second protein is not the GPCR protein.
Also provided by the invention is an antibody which is capable of binding a Drosophila melanogaster GPCR protein and which does not bind a GPCR protein of another species.
The invention also provides an isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, 3, 5 or 741 or a coding region thereof. In a specific embodiment the nucleic acid is DNA. The invention further provides an isolated RNA comprising the sequence of SEQ ID NO:1, 3, 5 or 741 wherein T (thymidine) residues are substituted by U (uracil) residues. Nucleic acids comprising a nucleotide sequence complementary to the nucleotide sequence of the foregoing nucleic acids are also provided.
The invention further provides an isolated nucleic acid comprising at least 50 contiguous nucleotides of SEQ ID NO:1, 3, 5 or 741.
Recombinant vectors comprising the above-described nucleic acids are also provided, as are recombinant host cells containing such a vector or a recombinant form of the nucleic acid.
The invention also provides a method for producing a Drosophila melanogaster GPCR protein comprising growing a recombinant cell containing the above-described vectors such that the GPCR protein encoded by said nucleic acid is expressed by the cell, and recovering the expressed GPCR protein. Also provided is the product of such method, said product being purified.
The invention further relates to a pharmaceutical composition comprising a therapeutically effective amount of a molecule comprising a fragment of a GPCR protein, said fragment (i) lacking an intracellular domain and a transmembrane domain, and (ii) exhibiting a functional activity of said GPCR protein; and a pharmaceutically acceptable carrier.
Another pharmaceutical composition provided by the invention comprises a therapeutically effective amount of an antibody that binds to the extracellular domain of a GPCR protein; and a pharmaceutically acceptable carrier. Alternatively, or additionally, the pharmaceutical composition comprises a therapeutically effective amount of a molecule comprising a nucleic acid encoding a fragment of a GPCR protein and a pharmaceutically acceptable carrier, said fragment (i) lacking an intracellular domain and a transmembrane domain, and (ii) exhibiting a functional activity of said GPCR protein.
The invention provides a purified Drosophila melanogaster protein which is encoded by a first nucleic acid that is hybridizable under high, moderate or low stringency conditions to a second nucleic acid, said second nucleic acid defined by the nucleotide sequence as set forth in SEQ ID NO:1, 3, 5 or 741.
The invention provides an isolated nucleic acid comprising a nucleotide sequence which encodes an amino acid sequence set forth in SEQ ID NO:2, 4, 6 or 32,370.
The invention provides a non-human transgenic animal which contains a recombinant nucleotide sequence that is inserted into or replaces at least a portion of the genomic sequence corresponding to the nucleotide sequence set forth in SEQ ID NO:1, 3, 5 or 741.
The invention provides a method for identifying a molecule that binds to a protein, said protein comprising an amino acid sequence depicted in SEQ ID NO:2, 4, 6 or 32,370, comprising contacting one or more candidate molecules with said protein under conditions conducive to binding to said protein; and detecting any binding that occurs of said candidate molecules to said protein.
The invention provides a method for screening for a molecule that modulates directly or indirectly the formation of a complex of a protein comprising an amino acid sequence depicted in SEQ ID NO:2, 4, 6 or 32,370 and its respective ligand comprising measuring the level of said complex formed under conditions conducive to formation of the complex, and comparing the levels of said complex with the levels of said complex that are formed in the absence of said molecule, wherein a higher or lower level of said complex in the presence of said molecule indicates that the molecule modulates formation of said complex.
The invention provides a method for screening for a molecule that modulates directly or indirectly the activity of a protein comprising an amino acid sequence depicted in SEQ ID NO:4 comprising measuring the potassium conductance of a cell expressing said protein in the presence of a candidate molecule, and comparing the levels of potassium conductance with the levels of potassium conductance in the absence of said molecule, wherein a higher or lower level of potassium conductance in the presence of said molecule indicates that the molecule modulates the potassium conductance of a cell expressing said protein.
The invention further provides a purified Drosophila melanogaster chitin synthase comprising or defined by the amino acid sequence set forth in SEQ ID NO:42,135, as well as a purified derivative of the chitin synthase, which is able to display one or more functional activities of a Drosophila melanogaster chitin synthase. In a specific embodiment, the derivative is able to be bound by an antibody directed against a Drosophila melanogaster chitin synthase. In another specific embodiment, the derivative is a fragment. The invention also provides a purified fragment of a Drosophila melanogaster chitin synthase protein, which fragment comprises a domain of the protein selected from the group consisting of the extracellular domain, the intracellular domain, a membrane spanning domain, and the catalytic domain. In a specific embodiment, the fragment comprises at least one membrane spanning domain.
The invention also provides a mature Drosophila melanogaster chitin synthase protein defined by the amino acid sequence set forth in SEQ ID NO:42,135 from which the secretory signal peptide sequence has been removed. Also provided is a purified protein comprising at least 25 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO:42,135; in a specific embodiment, such protein is fused to a second protein that is not a chitin synthase protein. The invention further relates to a chimeric protein comprising a fragment of a Drosophila melanogaster chitin synthase protein consisting of at least 25 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO:42,135 fused to a second protein, in which the second protein is not the GPCR protein.
The invention further provides an antibody which is capable of binding a chitin synthase protein as described above, and which does not bind another Drosophila melanogaster chitin synthase protein.
Also provided is an isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:10,540 or a coding region thereof. In a specific embodiment, the nucleic is DNA. The invention also relates to an isolated RNA comprising the sequence of SEQ ID NO:10,540 wherein T (thymidine) residues are substituted by U (uracil) residues. The invention further provides an isolated nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of one of the above-described nucleic acids. The invention also provides an isolated nucleic acid comprising at least 50 contiguous nucleotides of SEQ ID NO:10,540. A recombinant vector comprising an above-described nucleic acid, and a recombinant host cell containing the vector, are also provided. The invention further relates to a recombinant host cell containing an above-described nucleic acid, said nucleic acid being recombinant. The invention provides a method for producing a Drosophila melanogaster chitin synthase protein comprising growing a recombinant cell containing the above-described vector such that the chitin synthase protein encoded by said nucleic acid is expressed by the cell, and recovering the expressed chitin synthase protein; the purified product of such method is also provided. The invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a molecule comprising a fragment of the above-described chitin synthase protein, said fragment comprising the catalytic domain of the chitin synthase protein; and a pharmaceutically acceptable carrier. Also provided is a pharmaceutical composition comprising a therapeutically effective amount of an antibody that binds to the catalytic domain of the above-described chitin synthase protein; and a pharmaceutically acceptable carrier. The invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a molecule comprising a nucleic acid encoding a fragment of the above-described chitin synthase protein and a pharmaceutically acceptable carrier, said fragment comprising the catalytic domain of the chitin synthase protein.
The invention provides a purified Drosophila melanogaster protein which is encoded by a first nucleic acid that is hybridizable under high, moderate or low stringency conditions to a second nucleic acid, said second nucleic acid defined by the nucleotide sequence set forth in SEQ ID NO:10,540.
The invention also provides an isolated nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence set forth in SEQ ID NO:42,135.
The invention also provides a non-human transgenic animal which contains a recombinant nucleotide sequence that is inserted into or replaces at least a portion of the genomic sequence corresponding to the nucleotide sequence set forth in SEQ ID NO:10,540.
The invention also provides a method for identifying a molecule that binds to a protein, said protein comprising the amino acid sequence set forth in SEQ ID NO:42,135, comprising contacting one or more candidate molecules with said protein under conditions conducive to binding to said protein; and detecting any binding that occurs of said candidate molecules to said protein.
The invention also provides a method for screening for a molecule that modulates directly or indirectly the activity of a protein comprising the amino acid sequence depicted in SEQ ID NO:42,135 comprising measuring the chitin synthase activity of a cell expressing said protein in the presence of a candidate molecule, and comparing the levels of chitin synthase activity with the levels of chitin synthase activity in the absence of said molecule, wherein a higher or lower level of chitin synthase activity in the presence of said molecule indicates that the molecule modulates the chitin synthase activity of a cell expressing said protein.
The invention also provides a method for protecting a plant or animal against a pest comprising contacting the plant or animal with a pesticide formulation comprising (3-aminopropyl)methylphosphinic acid, and a carrier.
The invention also provides a purified protein comprising an amino acid sequence of any one amino acid sequence of SEQ ID NOS:31,636 to 46,852.
The invention also provides a purified protein comprising an amino acid sequence of any one amino acid sequence of SEQ ID NOS:46,853 to 62,485.
Also provided is a purified protein comprising an amino acid sequence of at least n contiguous amino acids of any one amino acid sequence of SEQ ID NOS:31,636 to 62,485, wherein for each respective SEQ ID NO, n is the number of amino acids listed in the column entitled xe2x80x9c100% identity lengthxe2x80x9d in Table 2. In particular embodiments, such purified protein comprises an amino acid sequence of least n+10 contiguous amino acids of any one amino acid sequence of SEQ ID NOS:31,636 to 62,485, wherein for each respective SEQ ID NO, n is the number of amino acids listed in the column entitled xe2x80x9c100% identity lengthxe2x80x9d in Table 2; or comprises an amino acid sequence of least n+50 contiguous amino acids of any one amino acid sequence of SEQ ID NOS:31,636 to 62,485, wherein for each respective SEQ ID NO, n is the number of amino acids listed in the column entitled xe2x80x9c100% identity lengthxe2x80x9d in Table 2.
The invention also provides a purified protein comprising an amino acid sequence having a BLAST score value of at least b when compared to any one amino acid sequence of SEQ ID NOS:31,636 to 62,485, wherein for each respective SEQ ID NO, b has a value greater than the BLAST score value listed in the column entitled xe2x80x9cBLAST scorexe2x80x9d in Table 2 for each respective SEQ ID NO, and wherein the BLAST score is determined using the same algorithm as used to calculate the BLAST score value of Table 2. In specific embodiments, for each respective SEQ ID NO, b has a value 3% greater than the BLAST score value listed in the column entitled xe2x80x9cBLAST scorexe2x80x9d in Table 2 for each respective SEQ ID NO; or for each respective SEQ ID NO, b has a value 10% greater than the BLAST score value listed in the column entitled xe2x80x9cBLAST scorexe2x80x9d in Table 2 for each respective SEQ ID NO.
The invention provides a purified derivative of one of the above-described proteins, which derivative is able to display one or more functional activities of said protein. In a specific embodiment, such derivative is able to be bound by an antibody directed against said protein.
The invention provides a purified fragment of a protein, said protein defined by an amino acid sequence of any one of SEQ ID NOS:31,636 to 62,485, said fragment comprising one or more domains of the protein as set forth in the column entitled xe2x80x9cPFam motifsxe2x80x9d or the column entitled xe2x80x9cProsite motifsxe2x80x9d for each respective SEQ ID NO in Table 2.
The invention provides a purified fragment of a protein, said fragment defined by an amino acid sequence of at least n contiguous amino acids of any one amino acid sequence of SEQ ID NOS:31,636 to 62,485, wherein for each respective SEQ ID NO, n is the number of amino acids listed in the column entitled xe2x80x9c100% identity lengthxe2x80x9d in Table 2. In specific embodiments, the above-described protein or fragment comprises a protein domain as set forth in the corresponding column entitled xe2x80x9cPFam motifsxe2x80x9d or the column entitled xe2x80x9cProsite motifsxe2x80x9d for each respective SEQ ID NO in Table 2.
The invention provides a purified fragment of a protein, said fragment defined by an amino acid sequence of at least n+50 contiguous amino acids of any one amino acid sequence of SEQ ID NOS:31,636 to 62,485, wherein for each respective SEQ ID NO, n is the number of amino acids listed in the column entitled xe2x80x9c100% identity lengthxe2x80x9d in Table 2. In specific embodiments, such fragment comprises a protein domain as set forth in the corresponding column entitled xe2x80x9cPFam motifsxe2x80x9d or the column entitled xe2x80x9cProsite motifsxe2x80x9d for each respective SEQ ID NO in Table 2.
The invention provides a purified fragment of a protein, said protein defined by an amino acid sequence having a BLAST score value of at least b when compared to any one amino acid sequence of SEQ ID NOS:31,636 to 62,485, wherein for each respective SEQ ID NO, b has a value greater than the BLAST score value listed in the column entitled xe2x80x9cBLAST scorexe2x80x9d in Table 2 for each respective SEQ ID NO, said fragment comprising a domain of the protein as set forth in the column entitled xe2x80x9cPFam motifsxe2x80x9d or the column entitled xe2x80x9cProsite motifsxe2x80x9d for each respective SEQ ID NO in Table 2, and wherein the BLAST score is determined using the same algorithm as used to calculate the BLAST score value of Table 2. In a specific embodiment, for each respective SEQ ID NO, b has a value 10% greater than the BLAST score value listed in the column entitled xe2x80x9cBLAST scorexe2x80x9d in Table 2 for each respective SEQ ID NO.
The invention provides an isolated nucleic acid comprising a nucleotide sequence of any one nucleotide sequence of SEQ ID NOS:7 to 15,288 or a coding region thereof.
The invention provides an isolated nucleic acid comprising a nucleotide sequence of any one nucleotide sequence of SEQ ID NOS:15,289 to 31,635 or a coding region thereof.
The invention provides an isolated nucleic acid comprising a nucleotide sequence complementary to any one nucleotide sequence of SEQ ID NOS:7 to 31,635.
The invention provides an isolated RNA molecule comprising a coding region of any one of SEQ ID NOS:7 to 31,635, wherein T (thymidine) residues are replaced with U (uracil) residues.
The invention provides an isolated nucleic acid comprising a nucleotide sequence of at least n contiguous nucleotides of any one nucleotide sequence of SEQ ID NOS:7 to 31,635, or a nucleotide sequence reverse complementary thereto, wherein for each respective SEQ ID NO, n is the number of nucleotides listed in the column entitled xe2x80x9c100% identity lengthxe2x80x9d in Table 1. In specific embodiments, such nucleic acid comprises a nucleotide sequence comprising at least n+25 contiguous nucleotides of any one nucleotide sequence of SEQ ID NOS:7 to 31,635, or a nucleotide sequence reverse complementary thereto, wherein for each respective SEQ ID NO, n is the number of nucleotides listed in the column entitled xe2x80x9c100% identity lengthxe2x80x9d in Table 1; or comprises a nucleotide sequence comprising at least n+50 contiguous nucleotides of any one nucleotide sequence of SEQ ID NOS:7 to 31,635, or a nucleotide sequence reverse complementary thereto, wherein for each respective SEQ ID NO, n is the number of nucleotides listed in the column entitled xe2x80x9c100% identity lengthxe2x80x9d in Table 1.
The invention provides an isolated first nucleic acid capable of hybridizing to a second nucleic acid consisting of any one nucleotide sequence of SEQ ID NOS:7 to 31,635, or a nucleotide sequence reverse complementary thereto, under hybridization condition x, wherein for each respective SEQ ID NO, x is the hybridization condition listed in the corresponding column entitled xe2x80x9chybriz. conditionsxe2x80x9d in Table 1. In a specific embodiment, the first nucleic acid encodes a protein (i) comprising a protein domain of the deduced protein sequence for the respective SEQ ID NO, as set forth in Table 1; or (ii) capable of being bound by an antibody to a protein defined by the deduced protein sequence for the respective SEQ ID NO, as set forth in Table 1.
The invention provides an isolated nucleic acid comprising a nucleotide sequence having at least z % sequence identity with any contiguous 125 bases of any one nucleotide sequence of SEQ ID NOS:7 to 31,635, or a nucleotide sequence reverse complementary thereto, wherein for each respective SEQ ID NO, z has a value greater than the value listed in the column entitled xe2x80x9c125 bp % identityxe2x80x9d in Table 1, and wherein the % sequence identity is determined using the same algorithm as used to calculate the % sequence identity value of Table 1. In specific embodiments, such nucleic acid comprises a nucleotide sequence having at least z % sequence identity with any contiguous 125 bases of any nucleotide sequence of SEQ ID NOS:7 to 31,635, or a nucleotide sequence reverse complementary thereto, wherein for each respective SEQ ID NO, z has a value 3% greater than the value listed in the column entitled xe2x80x9c125 bp % identityxe2x80x9d in Table 1, and wherein the % sequence identity is determined using the same algorithm as used to calculate the % sequence identity value of Table 1; or comprises a nucleotide sequence having at least z % sequence identity with any contiguous 125 bases of any nucleotide sequence of SEQ ID NOS:7 to 31,635, or a nucleotide sequence reverse complementary thereto, wherein for each respective SEQ ID NO, z has a value 10% greater than the value listed in the column entitled xe2x80x9c125 bp % identityxe2x80x9d in Table 1, and wherein the % sequence identity is determined using the same algorithm as used to calculate the % sequence identity value of Table 1.
The invention provides an isolated nucleic acid comprising a nucleotide sequence having at least w % sequence identity with any contiguous 275 bases of any nucleotide sequence of SEQ ID NOS:7 to 31,635, or a nucleotide sequence complementary thereto, wherein for each respective SEQ ID NO, w has a value greater than the value listed in the column entitled xe2x80x9c275 bp % identityxe2x80x9d in Table 1, and wherein the % sequence identity is determined using the same algorithm as used to calculate the % sequence identity value of Table 1. In specific embodiments, such nucleic acid comprises a nucleotide sequence having at least w % sequence identity with any contiguous 275 bases of any nucleotide sequence of SEQ ID NOS:7 to 31,635, or a nucleotide sequence complementary thereto, wherein for each respective SEQ ID NO, w has a value 3% greater than the value listed in the column entitled xe2x80x9c275 bp % identityxe2x80x9d in Table 1, and wherein the % sequence identity is determined using the same algorithm as used to calculate the % sequence identity value of Table 1; or comprises a nucleotide sequence having at least w % sequence identity with any contiguous 275 bases of any nucleotide sequence of SEQ ID NOS:7 to 31,635, or a nucleotide sequence complementary thereto, wherein for each respective SEQ ID NO, w has a value 10% greater than the value listed in the column entitled xe2x80x9c275 bp % identityxe2x80x9d in Table 1, and wherein the % sequence identity is determined using the same algorithm as used to calculate the % sequence identity value of Table 1.
The invention provides an isolated nucleic acid comprising a nucleotide sequence having a BLAST score value of at least b when compared to any one nucleotide sequence of SEQ ID NOS:7 to 31,635, or a nucleotide sequence reverse complementary thereto, wherein for each respective SEQ ID NO, b has a value greater than the BLAST score value listed in the column entitled xe2x80x9cBLAST scorexe2x80x9d in Table 1 for each respective SEQ ID NO, and wherein the BLAST score is determined using the same algorithm as used to calculate the BLAST score value of Table 1. In specific embodiments, for each respective SEQ ID NO, b has a value 3% greater than the BLAST score value listed in the column entitled xe2x80x9cBLAST scorexe2x80x9d in Table 1 for each respective SEQ ID NO; or b has a value 10% greater than the BLAST score value listed in the column entitled xe2x80x9cBLAST scorexe2x80x9d in Table 1 for each respective SEQ ID NO.
In specific embodiments, the above-described nucleic acids are DNA.
Isolated nucleic acids comprising a nucleotide sequence encoding an above-described protein are also provided.
The invention provides an isolated nucleic acid comprising a nucleotide sequence which is at least 65% similar over an at least 20 contiguous nucleotides to any one of the nucleotide sequences of SEQ ID NOS:7-31,635. The invention also provides an isolated nucleic acid comprising a nucleotide sequence which is at least 95% similar over an at least 20 contiguous nucleotides to any one of the nucleotide sequences of SEQ ID NOS:7-31,635. In specific embodiments, such nucleic acid encodes a protein comprising a domain of the protein encoded by any one of SEQ ID NOS:7-31,635 as set forth in the column entitled xe2x80x9cPFAM motifsxe2x80x9d or the column entitled xe2x80x9cProsite motifsxe2x80x9d for each respective SEQ ID NO in Table 2.
Recombinant vectors comprising the above-described nucleic acids, and recombinant host cell containing such vectors or containing the nucleic acid in recombinant form are also provided. The invention also provides a method for producing a protein comprising growing a recombinant cell containing such a vector such that the protein encoded by said nucleic acid is expressed by the cell, and recovering the expressed protein. The purified product of such method is also provided.
The invention provides an antibody which is capable of binding an amino acid sequence of any one of SEQ ID NOS:46,853 to 62,485.
The invention provides a transgenic non-human animal which contains a recombinant nucleotide sequence that is inserted into or replaces at least a portion of the genomic sequence corresponding to the nucleotide sequence set forth in any one of SEQ ID NOS:7 to 31,635.
The invention provides a computer readable medium having recorded thereon the amino acid sequence of any one of SEQ ID NOS:31,636 to 62,485 or a nucleotide sequence of any one of SEQ ID NOS:7 to 31,635.
The invention provides a computer readable medium having recorded thereon amino acid sequences comprising SEQ ID NOS:31,636 to 46,852, respectively, or nucleotide sequences comprising SEQ ID NOS:7-15,288, respectively.
The invention provides a method for identifying a molecule that binds to a protein, said protein comprising an amino acid sequence of any one of SEQ ID NOS:31,636 to 62,485, comprising contacting one or more candidate molecules with said protein under conditions conducive to binding to said protein; and detecting any binding that occurs of said candidate molecules to said protein.